Bienzyme Magnetic Nanobiocatalyst with Fe3+âTannic Acid Film for

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Biotechnology and Biological Transformations

A bi-enzyme magnetic nanobiocatalyst with Fe3+tannic acid film for one pot starch hydrolysis hongjie bian, baoting sun, Jiandong Cui, sizhu ren, Tao Lin, Yuxiao Feng, and Shiru Jia J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02097 • Publication Date (Web): 27 Jul 2018 Downloaded from http://pubs.acs.org on July 28, 2018

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Journal of Agricultural and Food Chemistry

1

A bi-enzyme magnetic nanobiocatalyst with Fe3+-tannic acid film

2

for one pot starch hydrolysis

3

Hongjie Bian*2 Baoting Sun1 Jiandong Cui*1,2

4

Shiru Jia*1

5

1

6

Fermentation Microbiology, Ministry of Education, Tianjin University of Science and

7

Technology, No 29, 13th, Avenue, Tianjin Economic and Technological Development Area

8

(TEDA), Tianjin 300457, P R China

9

2

Sizhu Ren1

Tao Lin2

Yuxiao Feng2

State Key Laboratory of Food Nutrition and Safety, Key Laboratory of Industrial

Research Center for Fermentation Engineering of Hebei, College of Bioscience and

10

Bioengineering, Hebei University of Science and Technology, 26 Yuxiang Street,

11

Shijiazhang 050000, P R China

12

*

13

E-mail

14

[email protected] (H. Bian)

Corresponding authors: address:

[email protected]

(J.

Cui);

[email protected]

15 16 17 18 19 20 21 22

1

ACS Paragon Plus Environment

(S.

Jia);


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23

Abstract

24

In this study, a novel co-immobilization biocatalyst for one pot starch hydrolysis was

25

prepared through shielding enzymes on the Fe3O4/SiO2 core-shell nanospheres by a

26

Fe3+-tannic acid (TA) film. In brief, alpha amylase and glucoamylase were covalently

27

immobilized on amino modified Fe3O4/SiO2 core-shell nanospheres using glutarldehyde

28

as linker. Then, a Fe3+-CA protective film was formed through the self-assembly of Fe3+

29

and TA coordination complex (Fe3+-TA@Fe3O4/SiO2-enzymes). The film acts a “coating”

30

to prevent the enzyme from denaturation and detachment, thus significantly improve its

31

structural and operational stability. Furthermore, the immobilization efficiency reached

32

90%, and the maximum activity recovery of α-amylase and glucoamylase was 87% and

33

85%, respectively. More important, the bi-enzyme magnetic nanobiocatalyst with

34

Fe3+-TA

35

Fe3+-TA@Fe3O4/SiO2-enzymes kept 55% of the original activity after reused for 9 cycles,

36

indicating outstanding reusability. However, the bi-enzyme magnetic nanobiocatalyst

37

without Fe3+-TA film maintained 28% of initial activity.

38

Keywords: Fe3+-TA film; α-amylase and glucoamylase; Co-immobilization; Starch

39

hydrolysis

film

could

be

simply

recovered

40 41 42 43

2


by

a

magnet.

The

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44

Introduction

45

As a natural macromolecule material, starch are widely used in food and pharmaceutical

46

industries. Generally, starch is hydrolyzed by α-amylase (EC 3.2.1.1) and glucoamylase

47

(EC 3.2.1.3) to become glucose in food industries. The hydrolysis of starch includes

48

liquefaction and saccharification. The liquefaction is the first step, where the starch is

49

hydrolyzed to soluble oligosaccharides by α-amylase. Subsequently, in saccharification

50

step, the resultant oligosaccharides are further hydrolyzed to glucose by glucoamylase.1,2

51

However, the major challenges in free enzymatic reactions are instability and difficult

52

recovery, which restricting their industrial application.3,4 Several years ago, stability,

53

activity and selectivity of enzymes could be improved by immobilizing enzymes on a

54

suitable support.5,6 Generally, the main reasons for the improvement of immobilized

55

enzymes performance include prevention of subunit dissociation and aggregation,

56

reduction of autolysis or proteolysis by proteases, rigidification of the enzyme structure,

57

and generation of favorable microenvironments.7-9 For example, α-amylase and

58

glucoamylase were immobilized individually on various solid carriers including

59

functionalized glass beads,10 polyaniline,11 ion exchange beads,12 and mesoporous

60

silica.13 Meanwhile, α-amylase and glucoamylase has been co-immobilized successfully,

61

and the co-immobilized enzymes exhibited high glucose production efficiency.1,14

62

Multistep enzymatic reactions can be combined into one step reaction by

63

co-immobilization. Furthermore, this process does not have to separate the intermediates,

64

and can decrease waste generation and the cost of procedure by recycle.2,15 However,

3



65

co-immobilized enzymes has several problems. For instance, the inactivation of the least

66

stable enzyme can result in abandoning of other enzymes. Moreover, the optimized

67

preparation conditions are difficult to obtain when enzymes are co-immobilized on the

68

same support using the same protocol.16,17 Furthermore, co-immobilization enzyme

69

particles might form increased clumps when they are recovered to reuse by centrifugation

70

and filtration. The large clumps will increase mass transfer limitations for starch,

71

causing reduction of starch conversion rate. Furthermore, in enzyme immobilization, if

72

the active center is oriented towards the support surface, it will be unavailable for the

73

substrate.18,19 Therefore, it is necessary to develop novel strategy to overcome these

74

problems.

75

Recent years, magnetic nanoparticles have become excellent carriers for enzyme

76

immobilization due to their biocompatibility, superparamagnetism, high specific surface

77

area, efficient Brownian motion in solution, and outstanding mechanical strength.

78

Immobilized α-amylase on Fe3O4 magnetic nanoparticles coated with gold could retain

79

60% of the enzyme activity after 10 cycles, and showed the superior conversion

80

efficiency of starch.20 Likewise, the immobilized α-amylase on the surface of

81

silica-coated modified magnetite nanoparticles was also exhibited excellent reusability.21

82

In addition, α-amylase, glucoamylase, and pullulanase were co-immobilized onto amino

83

functionalized Fe3O4 magnetic nanoparticles. The co-immobilization enzymes exhibited

84

improved thermostability, reusability, and catalytic rate for starch.2 Similarly,

85

glucoamylase and α-amylase were co-immobilized on chitosan beads entrapping Fe3O4

4


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particles for consecutive starch hydrolysis. The highest glucose production was obtained

87

using the bi-enzyme magnetic nanobiocatalyst at an optimum loading ratio between

88

α-amylase and glucoamylase.22 These reports showed that the magnetite (Fe3O4)

89

nanoparticles was suitable for co-immobilization of enzymes. However, the magnetic

90

nanoparticles can not protect the enzymes from denaturing stresses, interaction of the gas

91

bubbles with the enzyme,23and extreme environment.24 Recently, some efforts have been

92

carried out to solve these problems. For instance, lipase was immobilized on silica

93

nanosphere, and a porous silica layer around the immobilized lipase was formed to

94

protect from proteases degradation.25 Likewise, immobilized β-galactosidase on silica

95

nanosphere was protected in a soft organosilica layer from degradation of proteases.26 In

96

addition, immobilized penicillin G acylase (PGA) on the surface of cordierite honeycomb

97

ceramics was shielded in an organosilica layer. The protective organosilica shell

98

prevented the denaturation and detachment of the enzyme.27

99

Recent years, supramolecular metal-organic thin films have drawn wide attention due to

100

their diverse properties, and become convenient functional films on a diverse array of

101

substrates such as planar and particulate ones,28 viruses,29 and living cells.30 Therefore,

102

we wonder whether Fe3+-tannic acid (TA) film could also be used to protect the

103

immobilized enzymes. Generally, enzymes can be immobilized on a support highly

104

activated with glutaraldehyde (GA) by hydrophobic, anionic exchange and covalent.31,32

105

In this study, the amino-functionalization Fe3O4/silica core-shell nanospheres were

106

activated with GA, then, α-amylase and glucoamylase were co-immobilized on the

5



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107

surface of GA activated Fe3O4/silica core-shell nanospheres. Subsequently, a protective

108

Fe3+-TA film at the surface of co-immobilized α-amylase and glucoamylase on the

109

surface of co-immobilized enzymes was formed by self-assemble of coordination

110

complexes (Fig. 1). The mesoporous Fe3+-TA film played a “protective layer” to avoid

111

enzyme denaturation and detachment. Furthermore, covalent attachment of enzyme

112

molecules on Fe3O4/silica core-shell nanospheres gives higher stability. Therefore, the

113

bi-enzyme magnetic nanocatalyst with Fe3+-TA film is promising to use for one pot starch

114

hydrolysis.

115 116

Experimental section

117

Materials. Tannic acid, fluorescein isothiocyanate (FITC), sulforhodamine 101, soluble

118

starch, and ferric chloride hexahydrate (FeCl3·6H2O) were obtained from Tianjin

119

pharmaceutical Co., Ltd. (Tianjin, China). Aminopropyltriethoxysilane (APTES),

120

tetramethoxysilane (TMOS), α-amylase and glucoamylase were obtained from

121

Sigma-Aldrich.

122

Preparation of magnetite nanoparticles.

123

Fe3O4

124

modifications.33 0.01 M Ferric chloride hexahydrate and 0.09 M CH3COONa·3H2O were

125

mixed with 100 mL (CH2OH)2, and intensively agitated at 200 rpm for 30 min. The

126

mixture was sealed and heated at 200 °C in a Teflon-lined stainless-steel autoclave. After

127

8 h, the products were recovered by magnetic field. Subsequently, the products were

nanoparticles

were

synthesized

as

described

6


previously

with

some

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128

rinsed three times by ethanol, and dried.

129

Synthesis and amino-functionalization of Fe3O4/SiO2 nanoparticles.

130

The Fe3O4/SiO2 nanoparticles were synthesized by a modified sol-gel method.26 Fe3O4

131

(0.1 g) was mixed with ethanol, DI water and NH3·H2O, and the mixture was stirred for

132

30 min. Subsequently, TMOS (0.3 g) was mixed into the above solution. After 8 h, the

133

precipitates were recovered by magnetic field. Then, the products were rinsed three times

134

by ethanol, and dried. Subsequently, the Fe3O4/SiO2 nanoparticles were amino

135

functionalized by APTES silanization. 0.1 g Fe3O4/SiO2 nanoparticles were mixed with

136

isopropyl alcohol (100 mL) and 1.0 mL of APTES with nitrogen bubbling for 30 minutes.

137

And then, the mixture was agitated at 70 °C for 6 h, and the precipitates were recovered

138

by magnetic field, and rinsed three times by ethanol, and dried.

139

Enzymes

140

(Fe3O4/SiO2-enzymes). Firstly, 200 mg amino-functional Fe3O4/SiO2 nanoparticles were

141

activated by suspending them in GA solution (0.1%, v/v) for 60 min. Subsequently, the

142

GA activated Fe3O4/SiO2 nanoparticles were recovered by a magnet, and rinsed five

143

times by DI water, and dried. For co-immobilization of α-amylase and glucoamylase,

144

200 mg activated Fe3O4/SiO2 nanoparticles were added into 2 mL free enzyme mixtures

145

with α-amylase (50 U) and glucoamylase (50 U), the mixture was stirred at 25 °C. After

146

8 h, the products were separated by a magnet, and washed 3 times with DI water, and

147

dried prior to use.

148

Formation of Fe3+-TA film on Fe3O4/SiO2-enzymes (Fe3+-TA@Fe3O4/SiO2-enzymes).

immobilized

on

Fe3O4/SiO2

nanoparticles

7


activated

with

GA


149

For Fe3+-TA@Fe3O4/SiO2-enzyme composites, 100 mg Fe3O4/SiO2-enzymes were

150

suspended in Ferric chloride hexahydrate solution (10 mg/mL), and TA (40 mg/mL)

151

solutions were added to the suspension. After stirring 10 min, the resulting bi-enzyme

152

magnetic nanobiocatalyst with Fe3+-tannic acid film (Fe3+-TA@Fe3O4/SiO2-enzymes)

153

was recovered by magnetic field, and rinsed three times by DI water, and dried.

154

Characterization. Scanning electron microscope (SEM) and Transmission electron

155

microscope (TEM) was carried out by JEOL JSM6700 and JEOL JEM2100, respectively.

156

Nitrogen adsorption analysis were performed by a Beckman coulter SA3100 analyzer.

157

Fourier transform infrared (FT-IR) spectroscopy (Thermo Nicolet Corporation, Madison,

158

WI, scan range: 400-4000 cm-1) was used to determine chemical composition. Powder

159

X-ray diffraction (PXRD) (D/Max-2500 diffractometer, Shimadzu, Japan) was used to

160

study crystal structures. A magnetic property measure system (MPMS, Quantum design)

161

was used to determine magnetisation.

162

Labeled enzymes with FITC and sulforhodamine. α-amylase and glucoamylase were

163

added into FITC solution (50 mg/mL, FITC in acetone) and sulforhodamine 101 solution

164

for 3 min, respectively. The labeled enzymes were then co-immobilized on Fe3O4/SiO2

165

particles. Fluorescence detection was carried out by a confocal laser scanning

166

microscopy (CLSM) (Leica Camera AG, Germany). The laser provided excitation of

167

FITC and sulforhodamine 101 at 488 and 586 nm, and emitted fluorescent light was

168

detected at 545 and 605 nm, respectively.

169

Activity assay. α-amylase and glucoamylase activity was measured by using 1% soluble

8


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170

starch (w/v, pH 7) and 1% maltose (w/v, pH 4) as a substrate, respectively. For free

171

enzyme mixture, 20 µL of free enzyme mixture were mixed with 1 mL soluble starch at

172

60 °C. After 20 min, 100 µL HCl (1 M) was used to terminate the reaction.22 For

173

co-immobilized enzymes, 50 mg enzyme samples were added into soluble starch (1%,

174

w/v). The mixture was incubated at 60 °C for 20 min. Subsequently, enzyme samples

175

were recovered by magnetic field. The reducing sugar and glucose concentrations in the

176

supernatant was measured by the DNS method and a YSI biochemistry analyzer (YSI

177

2700 SELECT).34 For the free enzymes and co-immobilized enzymes, one unit (U) of

178

enzyme activity is defined as the amount of enzyme required to release 1 µmol of

179

reducing sugar (estimated as glucose) per minute at 60 °C and pH 6.

180

Optimum temperature and pH of free and co-immobilized enzymes. Optimum

181

temperature was investigated in the range of 40-75 °C using 1% soluble starch (w/v) as

182

substrate (0.01 M acetate buffer, pH 6). Optimum pH was examined in various pH

183

buffers (3.0-7.0) using 1% soluble starch (w/v) as substrate at 60 °C for the free enzyme

184

mixture and at 70 °C for the co-immobilized enzymes.

185

Stability of free and co-immobilized enzymes. The thermal stability of free enzyme

186

mixture, Fe3O4/SiO2-enzymes, and Fe3+-TA@Fe3O4/SiO2-enzymes was investigated by

187

incubating enzyme samples at 70 °C for different time interval (10-70 min), followed by

188

residual activity determination, respectively. The pH stability was measured by

189

incubating enzyme samples at different pH (3-11). In addition, the tolerance of free

190

enzyme

mixture,

Fe3O4/SiO2-enzymes,

and

Fe3+-TA@Fe3O4/SiO2-enzymes

9


to


191

denaturants was measured by incubating enzyme samples in different denaturants for 30

192

min. The storage time of free enzyme mixture, Fe3O4/SiO2-enzymes, and

193

Fe3+-TA@Fe3O4/SiO2-enzymes was studied by storing them at 25 °C for different time

194

interval (3-15 days), and the residual activities were calculated as percentage of the

195

initial activity. Also the reusability of the Fe3+-TA@Fe3O4/SiO2-enzymes and

196

Fe3O4/SiO2-enzymes were measured by reusing it nine times. One pot starch hydrolysis

197

reaction was carried out for 30 min at 60 °C with 20 mg/ml of soluble starch. After

198

reaction, the co-immobilized enzymes were recovered by magnetic field, rinsed three

199

times by DI water for next cycle.

200 201

Results and Discussion

202

Synthesis of the Fe3+-TA@Fe3O4/SiO2-enzyme composites. The preparation of the

203

Fe3+-TA@Fe3O4/SiO2-enzymes involved three steps (Figure 1). First, magnetic Fe3O4

204

nanoparticles were prepared, then the Fe3O4 nanoparticles was coated with a thin dense

205

silica layer to form Fe3O4/SiO2 nanospheres, followed by synthesis of the APTES

206

modified Fe3O4/SiO2 particles. Second, to make enzymes immobilized covalently onto

207

the modified Fe3O4/SiO2 particles, GA was used to link with both the amino groups of

208

the APTES modified Fe3O4/SiO2 particles and enzymes. Lastly, the immobilized

209

enzymes were added into TA and FeCl3 ·6H2O solution, leading to the formation of an

210

Fe3+-TA film at the surface of the immobilized enzyme. To confirm the formation of

211

covalent bonds, Fe3O4/SiO2 particles with GA treatment and Fe3O4/SiO2 particles

10


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212

without GA treatment were incubated in the free enzyme mixture for 8 h, respectively.

213

Then the resultant particles were separated by an magnet, and washed many times with

214

DI water. Subsequently, activity of the co-immobilized enzymes was determined. The

215

results showed that the Fe3O4/SiO2 particles with GA treatment exhibited activity

216

whereas Fe3O4/SiO2 particles without GA treatment did not display activity. The

217

maximum activity recovery of alpha amylase and glucoamylase was 87% and 85%,

218

respectively. This results demonstrated formation of covalent bonds between enzyme

219

and supports.

220

TEM

221

Fe3+-TA@Fe3O4/SiO2-enzyme particles were shown in Fig. 2. Fe3O4 nanoparticles were

222

uniform nanosphere with 200 nm (Fig. 2A). For Fe3O4/SiO2 nanospheres, it can clearly

223

see that Fe3O4 nanospheres (dark nanoparticles) were coated by a dense silica layer with

224

10

225

Fe3+-TA@Fe3O4/SiO2-enzymes exhibited double layers structure, a thin and loose

226

Fe3+-TA film with 5 nm thickness was clearly observed (Fig. 2C, 2D). This Fe3+-TA film

227

around Fe3O4/SiO2-enzymes could be further confirmed by the change of colors. Before

228

encapsulation of Fe3+-TA film, the color of Fe3O4/SiO2-enzyme particles was grey.

229

However, the color of the particles changed into brown after the formation of Fe3+-TA

230

film (Fig. 3). Besides, in Fig. 4, the absorption bands at 1478 and 1229 cm-1 arise from

231

the catechol ring vibration of TA (Fig. 4)35,36 The two characteristic peaks of Si-O-Si

232

antisymmetric stretching and FeO were observed at 1105 cm-1 and 591 cm-1,

images

nm

of

thickness

Fe3O4

(Fig.

nanospheres,

2B).

In

contrast

Fe3O4/SiO2

to

11


nanospheres,

Fe3O4/SiO2

and

nanospheres,


Page 12 of 38

233

respectively.22, 37 In addition, N-H bending and C-N stretching band (amide I) of enzyme

234

was observed at 1640-1660 cm-1 (1644 cm-1).38 Furthermore, to ascertain that α-amylase

235

and glucoamylase were indeed co-immobilized on Fe3+-TA@Fe3O4/SiO2-enzyme

236

nanospheres, before immobilization, α-amylase and glucoamylase were labeled by using

237

FITC

238

Fe3+-TA@Fe3O4/SiO2 particles with α-amylase exhibited green fluorescence (Fig. 5A)

239

while the Fe3+-TA@Fe3O4/SiO2 particles with glucoamylase exhibited red fluorescence

240

(Fig. 5B). The Fe3+-TA@Fe3O4/SiO2-enzyme particles(including α-amylase and

241

glucoamylase) displayed yellow fluorescence (Fig. 5C). The results further demonstrated

242

that

243

Fe3+-TA@Fe3O4/SiO2-enzyme nanospheres. EDS analysis showed the presence of Si

244

element and Ca element in the Fe3O4/SiO2-enzyme and Fe3+-TA@Fe3O4/SiO2-enzyme

245

(Fig.

246

Fe3+-TA@Fe3O4/SiO2-enzyme. As we known, α-amylases have one essential calcium ion.

247

Therefore,

248

Fe3+-TA@Fe3O4/SiO2-enzymes. Besides, XRD patterns exhibited that diffraction peaks

249

of Fe3+-TA@Fe3O4/SiO2-enzyme were consistent with Fe3O4 particles, indicating that

250

the Fe3O4 nanospheres were well maintained in the Fe3+-TA@Fe3O4/SiO2-enzyme (Fig.

251

6). Therefore, the Fe3+-TA@Fe3O4/SiO2-enzyme exhibited strong magnetization

252

saturation values (65.7 emu/g) (Fig. 7). In addition, nitrogen gas adsorption/desorption

253

analysis showed that the Fe3O4/SiO2-enzymes and Fe3+-TA@Fe3O4/SiO2-enzymes

and

sulforhodamine,

α-amylase

5E,

5F),

the

and

glucoamylase

indicating

result

respectively.

the

indicated

were

presence

that

The

of

results

showed

indeed

SiO2

α-amylases

12


co-immobilized

and

was

that

α-amylase in

immobilized

on

the

on

the

the

Page 13 of 38


254

exhibited mesoporous structure. The pore size distribution of the Fe3O4/SiO2-enzymes

255

showed narrow and sharp peak centered at 20 nm (Fig. 8). In contrast, the

256

Fe3+-TA@Fe3O4/SiO2-enzymes had the multiple level pore size distribution (a sharp

257

band centered at pore size of 80 nm and a broad band between 3 and 285 nm,

258

respectively) (Fig. 9), manifesting their large pore and loose structures. Furthermore,

259

alpha amylase and glucoamylase in bi-enzyme magnetic nanobiocatalyst still retained

260

high activity (92% of the original activity) after the formation of Fe3+-TA film.

261

Obviously, the thin and loose structure with large pore did not result in the significant

262

mass transfer limitation for substrates. In a word, we successfully synthesized the

263

Fe3+-TA@Fe3O4/SiO2-enzymes.

264

Optimization of reaction conditions

265

α-amylase/glucoamylase ratio is a key factor on the one pot starch hydrolysis. Therefore,

266

α-amylase/glucoamylase ratio in free enzyme mixture was optimized. The results

267

showed that the optimization activity of free enzyme mixture was obtained at 60 °C and

268

pH 6.0 when α-amylase/glucoamylase ratio was at 2:3. This ratio was also used for

269

co-immobilization enzymes. In addition, we examined optimum temperature for the free

270

enzyme mixture, Fe3O4/SiO2-enzymes, and Fe3+-TA@Fe3O4/SiO2-enzymes. The results

271

were shown in Figure 10A. The optimum temperature of free enzyme mixture was at

272

60 °C. The optimum temperature was consistent with free α-amylase. However, free

273

glucoamylase, individually immobilized α-amylase or glucoamylase on Fe3O4/SiO2

274

exhibited the maximum activity at 65 °C (data not shown). Furthermore, this optimum

13



Page 14 of 38

275

temperature was maintained at 65 °C after co-immobilization of enzymes. The improved

276

temperature can be due to the fact that formation of covalent binding increases the

277

rigidity of the enzyme structure, which protected the enzyme structure from deformation

278

by heat exchange.37,39 The optimum temperature was increased to 70 °C after Fe3+-TA

279

film

280

Fe3+-TA@Fe3O4/SiO2-enzyme still kept higher relative activity than free enzyme

281

mixture and Fe3O4/SiO2-enzyme when the temperature was increased to 75 °C. These

282

results could be due to the integrated effects from multipoint covalent bond and Fe3+-TA

283

films, which increased rigidity of enzyme conformation and retarded heat transfer.21,35

284

In addition, the optimum pH value of free enzyme mixture, Fe3O4/SiO2-enzymes, and

285

Fe3+-TA@Fe3O4/SiO2-enzymes was also studied. The optimum pH of free α-amylase

286

and glucoamylase was at 5.5 and 5.0, respectively (data not shown). However, it is found

287

that optimum pH values was at 6.0 for free enzyme mixture (Fig. 10 B). In contrast to

288

free enzyme mixture, the optimum pH value of all co-immobilized enzymes was at 7.0.

289

It was indicated that the formation of Fe3+-TA film did not affect microenvironment

290

around enzyme active site. Furthermore, compared with free enzyme mixture,

291

co-immobilized enzymes showed excellent adaptability in a wide pH range especially in

292

the alkaline range. The enhanced stability of co-immobilized enzymes in alkaline

293

condition was attribute to generating a rigid enzyme structure due to the multipoint

294

covalent fixation of enzyme with support.40,41 As a result, the pH tolerance of

295

Fe3O4/SiO2-enzyme and Fe3+-TA@Fe3O4/SiO2-enzyme increased.

was

formed

around

the

Fe3O4/SiO2-enzymes.

14


Furthermore,

the

Page 15 of 38


296

Stability of Fe3+-TA@Fe3O4/SiO2-enzyme. Immobilized enzymes with improved

297

stability and recoverable behavior are necessary in industrial applications. Therefore, we

298

tested the stability of Fe3+-TA@Fe3O4/SiO2-enzymes against high temperature, extreme

299

pH, and chemical denaturants. The results showed that Fe3+-TA@Fe3O4/SiO2-enzymes

300

exhibited more thermostability than free enzyme mixture and Fe3O4/SiO2-enzymes

301

(Fig.11 A). The Fe3+-TA@Fe3O4/SiO2-enzymes maintained more than 50% of original

302

activities after 1 h incubation at 70 °C, whereas free enzyme mixture and

303

Fe3O4/SiO2-enzymes only retained 20% and 30% of initial activities, respectively. The

304

increase of thermostability is consistent with previous reports.20,21 The enhanced

305

thermostability could be attributed to increase of enzyme rigidification by multiple

306

covalent immobilization and additional protection of Fe3+-TA film.24,42 Importantly, the

307

increased thermal stability of glucoamylase in Fe3+-TA@Fe3O4/SiO2-enzymes suggested

308

its potential for one pot starch hydrolysis.

309

In addition, pH stability of different enzyme samples were shown in Fig. 11B. All

310

enzyme forms had similarly characteristic at pH 3-11. However, compared to the free

311

enzyme mixture, co-immobilized enzymes exhibited increased resistance to extreme pH.

312

Especially,

313

Fe3O4/SiO2-enzymes. For instance, Fe3+-TA@Fe3O4/SiO2-enzymes retained 80% of its

314

original activity after 30 min incubation at pH 11. However, Fe3O4/SiO2-enzymes only

315

retained 60% of its original activity. A similar result was also obtained while evaluating

316

the

Fe3+-TA@Fe3O4/SiO2-enzymes

stability

of

multienzyme

system

showed

the

against

15


more

denaturants

stability

(Fig

than

11D).


Page 16 of 38

317

Fe3+-TA@Fe3O4/SiO2-enzymes maintained 75% of its original activity in 6 M urea for 1

318

h. However, free enzyme mixture and Fe3O4/SiO2-enzymes only maintained 14% and

319

48% of their original activities, respectively. The improved stability against extreme pH

320

and denaturants could be attributed to preventing changes in the enzyme confirmation by

321

covalent attachment between enzyme and Fe3O4/SiO2 as well as retarding acid/alkali

322

transfer and the denaturant corrosion by the Fe3+-TA films protection. Besides, compared

323

with Fe3O4/SiO2-enzymes and free enzyme mixture, the Fe3+-TA@Fe3O4/SiO2-enzymes

324

displayed excellent storage stability (Fig. 11C). Free enzyme mixture and

325

Fe3O4/SiO2-enzymes only maintained 25% and 70% of their original activities after 15

326

days respectively. However, Fe3+-TA@Fe3O4/SiO2-enzyme still maintained 85% of

327

original activity.

328

In

329

Fe3+-TA@Fe3O4/SiO2-enzymes for performing several consecutive operating cycles

330

using 20 mg/mL of soluble starch solution as the substrate. The results were shown in

331

Fig 12. Fe3+-TA@Fe3O4/SiO2-enzymes could be used at least up to 9 cycles in the

332

reaction mixture under the same reaction conditions. Both Fe3O4/SiO2-enzymes and

333

Fe3+-TA@Fe3O4/SiO2-enzymes displayed decreased activity with the increase of cycle

334

time up to 9 cycles. Fe3O4/SiO2-enzymes only retained 28% of its initial activity.

335

However, Fe3+-TA@Fe3O4/SiO2-enzymes still maintained 55% of its original activity,

336

indicating that the Fe3+-TA@Fe3O4/SiO2-enzymes had better reusability than

337

Fe3O4/SiO2-enzymes. Taken together, our results demonstrated that the presence of

addition,

we

evaluated

the

reusability

of

the

16


Fe3O4/SiO2-enzymes

and

Page 17 of 38


338

Fe3+-TA films on the surface of the co-immobilized enzymes provided a “coating” to

339

prevent enzyme from denaturation and detachment. Therefore, this “coating” is efficient

340

for improving the performances of co-immobilized enzymes.

341

In summary, a bi-enzyme magnetic nanobiocatalyst with Fe3+-TA film for one pot starch

342

hydrolysis was prepared by covalent immobilization of alpha amylase and glucoamylase

343

onto Fe3O4/silica core-shell nanospheres. In the system, the Fe3+-TA film can improve

344

the tolerance of the enzyme to denaturation conditions, including high temperature,

345

extreme

346

nanobiocatalyst with Fe3+-TA film exhibits excellent reusability during the multiple

347

cycles of starch hydrolysis, indicating much higher catalytic performance than

348

co-immobilized enzymes without Fe3+-TA film.

pH

value

and

denaturants.

Furthermore,

the

bi-enzyme

magnetic

349 350

Acknowledgements

351

This work is supported by the National Natural Science Foundation of China under the

352

grant number of 21676069, Dr. J. Cui also thanks supports from the Natural Science

353

Foundation of Hebei Province, China (project no. B2018208041), and Hundreds of

354

outstanding innovative talents in Hebei province (III) under the grant number of

355

SLRC2017036.

356 357 358

17



359 360

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480 481 482 483 484

23



Page 24 of 38

485 486

Figure legends

487

Figure 1 Schematic illustration of Fe3O4@silica core shell nanospheres with film for enzyme protection.

488 489 490 491 492 493 494

Fe3+-TA

Figure 2 TEM images of (A) Fe3O4, (B) Fe3O4/SiO2, and (C) Fe3O4/SiO2-enzymes and (D) Fe3+-TA@Fe3O4/SiO2-enzymes. Figure

3

Appearance

of

(A)

Fe3O4/SiO2-enzymes

and

(B)

Fe3O4/SiO2-enzymes

and

Fe3+-TA@Fe3O4/SiO2-enzymes. Figure

4

FT-IR

spectra

analysis

of


495

Figure 5 Confocal microscope images of (A) Fe3+-TA@Fe3O4/SiO2-α-amylase; (B)

496

Fe3+-TA@Fe3O4/SiO2-glucoamylase; (C, F) Fe3+-TA@Fe3O4/SiO2-enzymes; and

497

EDS patterns of (D) Fe3O4; (E) Fe3O4/SiO2.

498

Figure 6 PXRD of (A) Fe3+-TA@Fe3O4/SiO2-enzymes, and (B) standard Fe3O4.

499

Figure 7 Magnetisation curves of Fe3+-TA@Fe3O4/SiO2-enzymes.

500

Figure 8 N2 adsorption-desorption isotherms and pore size distribution curves of

501 502 503 504 505

Fe3O4/SiO2-enzymes. Figure 9 N2 adsorption-desorption isotherms and pore size distribution curves of Fe3+-TA@Fe3O4/SiO2-enzymes. Figure

10

Effects of

temperature

(A) and

pH


24


(B)

on

the

activity

of

Page 25 of 38


506

Figure

11

Stability

of

free

enzyme

mixture,

Fe3O4/SiO2-enzymes,

and

507

Fe3+-TA@Fe3O4/SiO2-enzymes. (A) thermostability, (B) pH-stability, (C) storage

508

stability, (D) stability against denaturants.

509

Figure 12 Reusability of Fe3O4/SiO2-enzymes and Fe3+-TA@Fe3O4/SiO2-enzymes.

510 511

25



Fig. 1

1


Page 26 of 38

Page 27 of 38


Fig. 2

2



Fig. 3

3


Page 28 of 38

Page 29 of 38


Fig. 4

4



Fig. 5

5


Page 30 of 38

Page 31 of 38


Fig. 6

6



Fig. 7

7


Page 32 of 38

Page 33 of 38


Fig. 8

8



Page 34 of 38

0.12

Quantity adsorbed (mmol/g)

5 4

d(V)/D(D)

0.10 0.08 0.06 0.04

3

0.02

0

50

100

150

200

250

300

Pore size (nm)

2 1 0 0.0

0.2

0.4

0.6

0.8

Relative pressure (P/P0) Fig. 9

9


1.0

Page 35 of 38


Fig. 10

10



Fig. 11

11


Page 36 of 38

Page 37 of 38


Fig. 12

12



TOC Graphic

13


Page 38 of 38

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